PROJECT SUMMARY - Research Project 3 Members of the filovirus genera Ebolavirus and Marburgvirus are HHS Tier 1 Category A priority pathogens that cause severe and often fatal hemorrhagic fever (HF) in humans and nonhuman primates (NHPs). There are presently no approved therapeutics for ebolavirus or marburgvirus infections, and a broad spectrum postexposure treatment that confers protection across all medically relevant filoviruses does not exist. Genevant currently is developing RNA interference (RNAi) therapeutics targeting clinically relevant ebolaviruses and marburgviruses. A key component of a RNAi therapeutic approach is the need for delivery technologies to ensure uptake of the small interfering RNA (siRNA). Genevant's N-acetylgalactosamine (GalNAc) conjugate and lipid nanoparticle (LNP) platforms stabilize siRNA and ensure effective delivery to disease sites and target cells that are the primary sites of infection for filoviruses. The goal of this research proposal is to generate a single broad spectrum siRNA-conjugate targeting the most clinically relevant ebolaviruses and marburgviruses. This therapeutic will then be used in a combinatorial treatment strategy with either fully human anti-filovirus monoclonal antibodies or the RNA polymerase inhibitors GS-5734 or favipiravir (T-705). This combinatorial strategy is intended to provide an effective and complementary treatment of filovirus infection that is more effective than each therapeutic alone. In order to generate anti-filoviral siRNA conjugates, work is proposed in two parts that will focus on optimizing the GalNAc ligand and siRNA components in parallel. To ensure potent delivery of anti-filoviral siRNAs, novel GalNAc ligands will be synthesized and assessed for binding avidity and ranked for their ability to mediate RNA silencing (Aim 1). The most effective ligand-conjugate delivery platform will then be selected for conjugation to anti-filoviral siRNAs selected in Aim 2. Lead siRNAs, delivered via LNP, have already been identified with demonstrated efficacy against EBOV, SUDV, BDBV, MARV, and RAVV in our current U19 CETR grant. These anti-filoviral parental siRNA sequences will undergo chemical modification to enable compatibility with a ligand conjugate delivery platform. Chemically modified siRNAs retaining silencing activity against each clinically relevant filovirus will then be combined with the most effective ligands identified in Aim 1 and assessed for broad spectrum efficacy in NHP models of filovirus infection. Different combinations of siRNA-conjugates, monoclonal antibodies, and direct acting antiviral small molecules will be tested in filovirus- infected NHPs in Aim 3, with the goal of determining whether any of these combinatorial approaches will yield benefit in improving protection during late stages of disease.